The introduction of the polymerase chain reaction (PCR) and subsequent alternative amplification systems for nucleic acids has made it possible to use this genetic material as an examination material for diagnostic tests. This opens up new analytical opportunities especially for diagnosing hereditary diseases, predisposition for certain diseases and infectious diseases which among others also allow an early detection of the condition.
In order to convert the genetic material into a suitable form for enzymatic amplification, it is necessary to release it from the biological material. Furthermore, the nucleic acid must be protected from degradation by nucleases from the biological material or from the environment and also protected from degradation by chemical reaction conditions. The highest demands are made on the freedom from contamination of the biological sample and of the nucleic acid isolated therefrom. The nucleic acid should be present for the amplification in a buffered, aqueous, substantially salt-free solution.
Whereas very small amounts of analyte are basically used in PCR (pg-ng range), special problems require the processing of a larger amount of sample. In order to for example identify circulating tumour cells with a sensitivity of one tumour cell in a background of normal cells, the nucleic acid must for example be isolated from 10-20 ml of a blood sample. Then after homogenising the sample, an aliquot of the isolated RNA can be examined for the expression of a tumour-associated gene.
In addition to the classical methods of nucleic acid isolation by means of enzymatic, mechanical or chemical lysis of the sample material, subsequent extraction of the proteins and lipids by phenol and phenol/CHCl3 and precipitation of the nucleic acid from the aqueous phase using ethanol or i-propanol (Sambrook, J., et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989, 2nd Edition, 9.16-9.23; Ausubel, F. M., et al., Current Protocols in Molecular Biology, John Wiley & Sons, 1987, 2.1.1-2.4.5), some commercial kits have been developed in recent years especially for PCR sample preparation which utilize the property of nucleic acids that has been known since the end of the seventies of binding under chaotropic salt conditions to glass surfaces (Vogelstein, B., et al., Proc. Natl. Acad. Sci. USA 76 (1979) 615-619). Other components of biological material such as proteins, lipids or salts are not bound and therefore separated. Centrifugation vessels with inserted glass fleeces or silica gel suspensions which allow a batch process are known. Furthermore multiple devices in a strip and 96-well microwell plate format with glass fleeces recessed into the bottom are known and can be operated with the aid of a vacuum chamber attached underneath as well as by centrifugation. In these methods the volume of the samples is often limited. Furthermore, large amounts of buffer are necessary for an effective elution of the nucleic acids from the glass fleeces which results in a diluted solution of the isolated molecules and requires additional preparation steps for certain applications.
A modified method (Miller et al., Nucl. Acids. Res. 16:1215) uses a concentrated salt solution to precipitate proteins and other accompanying substances after the sample material has been lysed. The nucleic acids located in the supernatant are then precipitated by ethanol and collected by centrifugation. After the nucleic acids have been dissolved they can be used for amplification.
WO 93/11221 discloses a method and a device for isolating and purifying nucleic acids which uses anion exchangers and mineral carrier substances. WO 98/32877 discloses a device for isolating nucleic acids which is composed of two vessels which are connected by a closure element in which a material for binding nucleic acids is introduced. U.S. Pat. No. 4,956,298 discloses a separation or reaction column consisting of a centrifugation vessel and a receiving body wherein the receiving vessel contains a column material and the centrifugation vessel collects the discharge from the receiving body. DE 19512361 discloses a method for isolating a biological material which uses a compressible porous matrix for binding the biological material and uses the compression of the material to elute the material. EP 588564 describes a device for affinity separation comprising a capture membrane arranged in a pipette tip. WO 96/41810 discloses the withdrawal of DNA from a cell suspension with the aid of a hollow membrane filter and an ion exchange step. The production of a device containing a material for binding nucleic acids is known from EP 738733. The German utility model DE 298 03 712 U1 describes a device for treating biomolecules comprising a separation column which has a separating device and a collection vessel for the discharged liquid. In this device the funnel-shaped extended separating device also has (a) a volume for receiving a large amount of lysate and (b) a filter element (glass filter) which serves to adsorb nucleic acids from the lysate. In each centrifugation step the separating device is always used inserted into a centrifugation vessel. The centrifugation vessel is typically a 50 ml standard centrifuge vessel, for example a Falcon tube. A collecting vessel (microliter vessel) is plugged onto the bottom of the separating device in order to elute the adsorbed nucleic acids from the filter element. The separating device and attached collecting vessel are inserted into the centrifugation vessel whereby the collecting vessel touches the bottom of the centrifugation vessel. WO 2005/090567 describes a device consisting of a funnel element and a commercial spin column (also known as a “mini-spin column”; also referred to as a “separating column” in the following) with a glass fleece. An example of such a separating column is a HIGH PURE column (Roche Diagnostics Operations, Inc.). The funnel element has a volume for receiving a large amount of lysate. The bottom of the funnel element has an opening into which a spin column is inserted flush and sealed at the edge. This combination is inserted into a 50 ml standard centrifuge vessel for the first centrifugation step in which the centrifugal force presses the lysate through the glass fleece, the nucleic acids adsorb to the glass fleece and the discharge is collected in the centrifugation vessel. Subsequently the separating column can be removed from the funnel element and inserted into a microliter vessel in order to carry out further steps (e.g., washing steps, elution) with smaller centrifuges with a higher holding capacity and higher rotation speeds. Such a procedure can offer advantages with regard to processing speed and throughput. WO 2006/008085 shows an arrangement in which a separating column is plugged onto a funnel element without further attaching means.